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Carmen Buchrieser and Hubert Hilbi (eds.), Legionella: Methods and Protocols, Methods in Molecular Biology, vol. 954,DOI 10.1007/978-1-62703-161-5_18, © Springer Science+Business Media New York 2013

Chapter 18

Pathogen Vacuole Puri fi cation from Legionella -Infected Amoeba and Macrophages

Christine Hoffmann , Ivo Finsel , and Hubert Hilbi

Abstract

Legionella pneumophila replicates intracellularly in environmental and immune phagocytes within a unique membrane-bound compartment, the Legionella -containing vacuole (LCV). Formation of LCVs is strictly dependent on the Icm/Dot type IV secretion system and the translocation of “effector” proteins into the cell. Some effector proteins decorate the LCV membrane and subvert host cell vesicle traf fi cking pathways. Here we describe a method to purify intact LCVs from Dictyostelium discoideum amoebae and RAW 264.7 murine macrophages. The method comprises a two-step protocol: fi rst, LCVs are enriched by immuno-magnetic separation using an antibody against a bacterial effector protein speci fi cally localizing to the LCV membrane, and second, the LCVs are further puri fi ed by density gradient centrifugation. The puri fi ed LCVs can be characterized by proteomics and other biochemical approaches.

Key words: Amoeba , Dictyostelium discoideum , Density gradient centrifugation , Immuno-magnetic separation , Legionella pneumophila , Macrophage , Pathogen vacuole , Type IV secretion

Abbreviations

ACES N -(2-Acetamido)-2-aminoethanesulfonic acid GFP Green fl uorescent protein HEPES N -2-Hydroxy-ethylpiperazine- N ¢ -2-ethanesulfonic acid Icm/Dot Intracellular multiplication/defective organelle traf fi cking T4SS Type IV secretion system

310 C. Hoffmann et al.

A large number of pathogenic bacteria invade and replicate within host cells by forming distinct vacuolar compartments. Legionella pneumophila , the causative agent of a severe pneumonia termed Legionnaires’ disease, is a model organism for studying these vacu-olar compartments ( 1 ) . L. pneumophila is a Gram-negative envi-ronmental bacterium able to grow in extracellular but also intracellular niches. The intracellular niches are protozoa, includ-ing amoebae and ciliates, but also human macrophages ( 2 ) . Upon uptake by these phagocytes, L. pneumophila grows in a mechanisti-cally similar way in amoebae or macrophages within a unique membrane-bound compartment termed the Legionella -containing vacuole (LCV). To establish an LCV and to communicate with host cell compartments such as endosomes, endoplasmic reticulum (ER), mitochondria, and the Golgi apparatus, the bacteria use the Icm/Dot type IV secretion system to translocate as many as 275 different effector proteins into the host cell ( 3 ) . The manipulation of host functions and the recruitment of host factors to the LCV membrane promote bacterial survival and formation of the intrac-ellular replication-permissive niche of L. pneumophila ( 4 ) .

The biogenesis of particle- or pathogen-containing vacuoles has been investigated by proteomic analysis of isolated compartments using 2-D gel electrophoresis or liquid chromatography coupled to mass-spectrometry. Subcellular vesicles isolated from the social soil amoeba Dictyostelium discoideum or from mammalian phago-cytes include latex bead phagosomes ( 5– 7 ) , or vacuoles contain-ing intracellular microorganisms, such as Leishmania parasites ( 8 ) , Listeria innocua ( 9 ) , Mycobacterium avium ( 10 ) , Rhodococcus equi ( 11 ) , Salmonella enterica serovar Typhimurium ( 12 ) , or L. pneumophila ( 13 ) .

Yet, the puri fi cation procedures employed in these studies are time-consuming and tedious, e.g., they require electron-micros-copy to determine vacuole integrity and purity. Furthermore, these protocols are based solely on density gradient centrifugation using sucrose or iodinated materials such as Optiprep or Histodenz/Nycodenz, and they do not exploit speci fi c molecular features of the pathogen-containing vacuoles to be enriched. As a conse-quence, the discrimination between relevant vacuole components and co-puri fi ed irrelevant proteins has been dif fi cult.

To overcome these limitations, we established a straightfor-ward protocol to isolate intact LCVs from D. discoideum using an L. pneumophila LCV marker, which exclusively binds to the LCV membrane ( 14, 15 ) . Speci fi cally, we targeted the Icm/Dot sub-strate SidC, which anchors to the LCV membrane by binding to the host cell lipid phosphatidylinositol 4-phosphate (PtdIns(4) P ),

1. Introduction

1.1. Formation of the Legionella -Containing Vacuole

1.2. Puri fi cation of Pathogen Vacuoles

31118 Pathogen Vacuole Purifi cation from Legionella-Infected Amoeba and Macrophages

with an af fi nity-puri fi ed antibody ( 16, 17 ) . The immuno-af fi nity step was followed by classical Histodenz density gradient centrifu-gation. A proteomics analysis of LCVs puri fi ed by this method revealed 566 host proteins, including 60 proteins localizing to phagocytic vesicles and 18 proteins associated with the ER or Golgi apparatus, as well as a number of small GTPases involved in endo-somal and secretory vesicle traf fi cking, which have not been impli-cated in LCV formation before ( 18 ) .

Here, we describe an LCV puri fi cation protocol based on immuno-magnetic separation followed by density gradient cen-trifugation that is suitable to enrich LCVs from infected D. discoi-deum amoeba, as well as from murine RAW 264.7 macrophage-like cells (Fig. 1 ). The protocol allows monitoring the enrichment of LCVs readily by light microscopy using fl uorescently labeled L. pneumophila and phagocytes (e.g., D. discoideum producing calnexin-GFP). Moreover, the puri fi ed LCVs can be directly ana-lyzed by mass-spectrometry-based comparative proteomics, Western blot, or immune- fl uorescence microscopy.

Use deionized, distilled water in all recipes and protocol steps.

1. We use L. pneumophila strain Philadelphia 1 producing the red fl uorescent protein DsRed-Express encoded by plasmid pSW001 ( 14, 19 ) . More than 90% of the bacteria produce DsRed in the post-exponential (virulent) growth phase.

2. Materials

2.1. Legionella pneumophila

Fig. 1. Schematic overview of the ( a ) enrichment of LCVs by immuno-magnetic separation and ( b ) puri fi cation of LCVs by Histodenz density gradient centrifugation.

312 C. Hoffmann et al.

2. ACES yeast extract (AYE) medium ( 20 ) : 10 g/L N -(2-acetamido)-2-aminoethane-sulfonic acid (ACES), 10 g/L Bacto™ yeast extract (Difco; see Note 1), 3.3 mM L -cysteine, 0.6 mM Fe(NO 3 ) 3 . Add 10 g of ACES and 10 g of yeast extract in 950 mL of H 2 O. Add fi lter-sterilized 0.4 g/10 mL L -cysteine and 0.25 g/10 mL Fe(NO 3 ) 3 solutions (see Note 2). Adjust the pH to 6.9 with 10 M KOH. To select for plasmid pSW001 add 5 m g/L chloramphenicol (Cam, stock: 30 mg/mL etha-nol). Pass the medium several times through a glass fi ber fi lter paper, followed by a 0.2 m m fi lter cartouche. Store the medium at 4°C in the dark (see Note 3).

3. Charcoal yeast extract (CYE) agar plates ( 21 ) : 10 g/L ACES, 10 g/L Bacto™ yeast extract (Difco; see Note 1), 2 g/L acti-vated charcoal powder (puriss. p.a.), 15 g/L agar, 3.3 mM L -cysteine, 0.6 mM Fe(NO 3 ) 3 . Dissolve 10 g of ACES and 10 g of yeast extract in 950 mL of H 2 O and adjust the pH to 6.9 with 10 M KOH. Transfer the solution to a 1 L Schott bottle containing 2 g of activated charcoal powder, 15 g of agar, and a stir bar. Autoclave and let the agar solution cool down to 50°C. Add fi lter-sterilized 0.4 g/10 mL L -cysteine and 0.25 g/10 mL Fe(NO 3 ) 3 solutions (see Note 2). To select for plasmid pSW001 add 5 m g/L Cam. Mix the solution on a magnetic stirrer and pour plates. Let the plates dry for 1 day at room temperature and store at 4°C for up to 6 months.

4. Chloramphenicol: Stock concentration 30 mg/mL in ethanol, fi lter-sterilize fi aliquots, and store at −20°C.

1. We use the wild-type D. discoideum strain AX3 harboring a plasmid encoding calnexin-GFP ( 22 ) (see Note 4).

2. HL5 medium, modi fi ed ( 23 ) : 5 g/L BBL™ yeast extract (Becton Dickinson; see Note 1), 5 g/L Bacto™ Proteose Peptone (Becton Dickinson; see Note 1), 5 g/L BBL™ Thiotone™ Peptone (Becton Dickinson; see Note 1), 11 g/L D(+)glucose monohydrate (see Note 5), 2.5 mM Na 2 HPO 4 , 2.5 mM KH 2 PO 4 . Adjust the pH with 1 M KOH or 1 M HCl to 6.5 ± 0.1, and add 5–20 m g/mL geneticin (G418) to select for the plasmid. Autoclave and store the medium at 4°C for up to 1 year. If necessary, add Penicillin/Streptomycin (Pen/Strep) or Fungizone to maintain sterility. The modi fi ed HL5 medium supports axenic growth of D. discoideum .

3. Sörensen phosphate buffer ( 24 ) with CaCl 2 (SorC): 2 mM Na 2 HPO 4 , 15 mM KH 2 PO 4 , 50 m M CaCl 2 . Adjust the pH with 1 M KOH or 1 M HCl to 6.0 ± 0.1, autoclave, and store at room temperature for up to 2 years. Add 1 mL of a sterile 50 mM CaCl 2 stock solution. SorC Buffer is used as a washing and fl ow cytometry buffer.

2.2. Dictyostelium discoideum

31318 Pathogen Vacuole Purifi cation from Legionella-Infected Amoeba and Macrophages

4. Antibiotics for cell culture: 5–20 m g/mL G418 (stock: 20 mg/mL H 2 O), 100 U/mL Pen (stock: 10,000 U/mL), 0.1 mg/mL Strep (stock: 10 mg/mL H 2 O). Filter-sterilize and store the stock solutions at −20°C for up to 2 years.

1. We use the mouse leukemic monocyte macrophage cell line RAW 264.7.

2. RPMI 1640 medium: Supplement the commercially available RPMI 1640 medium with 2 mM L -glutamine and 10% heat-inactivated fetal calf serum (FCS). If necessary, add 100 U/mL Pen and 0.1 mg/mL Strep to maintain sterile conditions.

3. Phosphate-buffered saline (PBS) buffer: 1.37 M NaCl, 26.8 mM KCl, 14.7 mM KH 2 PO 4 , 78.1 mM Na 2 PO 4 .

1. HS (homogenization buffer): 20 mM N -2-hydroxy-ethylpiperazine- N ¢ -2-ethanesulfonic acid (HEPES), 250 mM sucrose, 0.5 mM ethyleneglycoltetraacetic acid (EGTA). Adjust the pH to 7.2 with 1 M KOH, fi using a 0.2 m m fi lter (do not autoclave), and store at 4°C for up to 6 months. Freshly add a complete EDTA-free protease inhibitor cocktail tablet (Roche, cat. no. 11-836-170-001) according to the manufacturer’s instructions (1 tablet/10 mL solution), and use the buffer ice cold.

2. Blocking reagent: Fetal calf serum (FCS) or calf-serum (CS) at a fi nal concentration of 2% (v/v).

3. 10 and 35% Histodenz solutions: Dissolve 10 g or 35 g Histodenz (Sigma-Aldrich) in PBS to an end volume of 100 mL. Store at 4°C in the dark for up to 2 years.

4. Primary antibody: Af fi nity-puri fi ed polyclonal rabbit anti-SidC serum (NeoMPS, Strasbourg, France).

5. Secondary antibody: Magnet-activated cell sorting (MACS) goat anti-rabbit IgG micro beads (Miltenyi Biotec, cat. no. 130-048-602).

6. Sterile 0.01% poly- L -lysine solution. 7. 4% (w/v) paraformaldehyde (PFA): Wear gloves and mask and

weigh 40 g PFA in a chemical fume hood. Dissolve in 1 L PBS, while constantly stirring and heating the solution to 50°C. Adjust the pH to 7.3. Store at −20°C for up to 2 years.

1. Incubators (23°C, 25°C, 37°C, 37°C with 5% CO 2 ) with rota-tion wheel or shaker (37°C).

2. Overhead rotation wheel (at 4°C). 3. Stainless steel ball homogenizer (8 m m clearance, 0.5 mL

chamber; Isobiotec, Germany). 4. Spectrophotometer.

2.3. RAW 264.7 Macrophages

2.4. Cell Lysis and LCV Puri fi cation

2.5. Equipment and Consumption Items

314 C. Hoffmann et al.

5. MACS separator (e.g., MACS multistand, Miltenyi Biotec). 6. MACS-MS columns (Miltenyi Biotec, cat. no. 130-042-201). 7. 75 cm² tissue culture fl asks. 8. 15 mL test tubes, 1.5 mL microcentrifuge tubes. 9. Plastic cell-scraper, 3 mL plastic Luer-lock syringes. 10. Glass Pasteur pipettes (230 mm, unplugged).

1. Growth on CYE agar or in AYE medium: Streak out L. pneu-mophila from frozen glycerol stocks onto CYE plates contain-ing 5 m g/mL Cam to maintain plasmid pSW001, and grow for 3 days at 37°C. Inoculate in a 15 mL test tube 3 mL AYE medium containing 5 m g/mL Cam with 0.1 mL of a L. pneu-mophila suspension (OD 600 = 0.1) and incubate on a rotation wheel (approximately 100 rpm) for 21–22 h at 37°C, until bacteria reach their peak infectivity ( fi nal OD 600 ³ 3.0) (see Note 6). As a control for L. pneumophila viability, plate 20 m L of a 10 5 /mL bacterial solution on CYE agar plates.

1. Cultivate D. discoideum in 10 mL HL5 medium in 75 cm 2 tis-sue culture fl asks at 23°C with 20 m g/mL G418 to maintain the plasmid encoding calnexin-GFP. Split the culture two to three times a week by tapping off the amoebae (see Note 7).

1. Cultivate RAW 264.7 macrophages in RPMI 1640 medium supplemented with 2 mM L -glutamine and 10% heat-inacti-vated FCS in 75 cm 2 tissue culture fl asks at 37°C and 5% CO 2 . Split the culture two times a week using a plastic cell scraper.

1. Seed 1 × 10 7 D. discoideum or RAW 264.7 macrophages in a 75 cm 2 tissue culture fl ask and grow without antibiotic over-night in 10 mL HL5 at 23°C ( D. discoideum ) or in 10 mL RPMI 1640 at 37°C (RAW 264.7 macrophages) (see Note 8). Use up to three fl asks per infection and sample (total of 6 × 10 7 host cells).

2. Add 500 m L of L. pneumophila culture (see Subheading 3.1 ) per 75 cm 2 tissue culture fl ask and swirl gently. The multiplicity of infection (MOI) is approximately 50 (see Note 9).

3. Centrifuge the fl ask at 500 × g for 10 min at 25°C to synchro-nize the infection.

4. Incubate for 1 h at 25°C ( D. discoideum ) or 37°C (RAW 264.7 macrophages) (see Note 10).

3. Methods

3.1. Handling of L. pneumophila

3.2. Handling of D. discoideum

3.3. Handling of RAW 264.7 Macrophages

3.4. Infection of Phagocytes with L. pneumophila

31518 Pathogen Vacuole Purifi cation from Legionella-Infected Amoeba and Macrophages

1. After an incubation period of 1 h, place the fl asks on ice, and wash the cells two to three times with 10 mL cold SorC ( D. discoideum ) or PBS (RAW 264.7 macrophages), respec-tively, to remove medium and non-phagocytosed bacteria. Perform the washing steps by gently agitating at 25°C ( D. discoideum ) or 37°C (RAW 264.7 macrophages).

2. Resuspend up to 2 × 10 7 infected phagocytes in 3 mL ice-cold HS buffer containing Roche complete EDTA-free protease inhibitor using a plastic cell scraper. Transfer the suspension into a 3 mL disposable Luer-lock syringe. Set aside 180 m L for analysis by fl uorescence microscopy (see Note 11).

3. Wash the ball homogenizer (8 m m exclusion size) thoroughly with distilled water before use to avoid any detergent contami-nation, and fl ush the prechilled homogenizer with cold HS buffer to get rid of any air bubbles.

4. Mount the syringe containing the suspension of L. pneumophila -infected phagocytes, and press the suspension through the homogenizer into a second syringe. Passage the suspension back and forth seven to nine times, until the homogenate becomes clear. Keep the homogenate on ice and set aside 180 m L homo-genate for fl uorescence microscopy (see Note 12).

5. Before proceeding with a different sample, dismantle and thor-oughly wash the ball homogenizer again.

1. Pool the matching samples of cell homogenates (~9 mL per 6 × 10 7 cells) into a 15 mL plastic tube.

2. Add FCS or CS as blocking reagent to a fi nal concentration of 2% (v/v), and incubate for 30 min on an overhead spinning wheel at 10–20 rpm (4°C) (see Note 13).

3. Use a primary antibody directed against a bacterial marker speci fi cally binding to LCVs. Vortex the antibody solution prior to use and incubate the solution with the homogenate of L. pneumophila -infected phagocytes on an overhead spinning wheel at 10–20 rpm for 1 h (4°C). Af fi nity-puri fi ed polyclonal rabbit antiserum directed against the L. pneumophila effector protein SidC ( 25, 26 ) is used at a dilution of 1:3,000 (see Note 14).

4. Centrifuge the homogenate at 2,700 × g for 15 min (4°C). Remove the supernatant completely, resuspend the pellet in 1.5 mL HS buffer per homogenate of 6 × 10 7 phagocytes, and transfer the suspension to a new 15 mL centrifuge tube.

5. Use a secondary antibody coupled to MACS micro-beads. Vortex the antibody suspension prior to use and add appropri-ate amounts to the homogenate. Goat anti-rabbit IgG MACS micro-beads are used against the polyclonal rabbit anti-SidC serum at a concentration of 20 m L magnetic bead slurry per

3.5. Homogenization of Infected Phagocytes

3.6. Puri fi cation of LCVs by Immuno-Magnetic Separation

316 C. Hoffmann et al.

0.5 mL concentrated cell homogenate. Incubate for 30 min on an overhead spinning wheel at 10–20 rpm (4°C).

6. Place a magnetic MACS-MS separation column into an MACS separator and equilibrate with 0.5 mL ice-cold HS buffer by passing through the column by gravity fl ow.

7. Load the suspension of antibody-treated homogenate (1.5 mL, corresponding to 6 × 10 7 phagocytes) onto an equilibrated MACS-MS column by gravity fl ow. Set aside 30 m L of the anti-body-treated homogenate for fl uorescence microscopy.

8. Wash the loaded MACS columns three times with 0.5 mL ice-cold HS buffer by gravity fl ow. Set aside 30 m L of the fl ow-through for fl uorescence microscopy (Fig. 2 ).

Fig. 2. Immuno-magnetic separation of Legionella -containing vacuoles from D. discoideum and RAW 264.7 macrophages. The images show the fl ow-through and eluate of a magnetic cell separation (MACS) column loaded with homogenate of D. discoideum or RAW 264.7 macrophages infected with DsRed-producing L. pneumophila ( red ). ( a ) Flow-through and ( b ) eluate of Legionella -infected calnexin-GFP producing D. discoideum ( green ). ( c ) Flow-through and ( d ) eluate of Legionella -infected RAW 264.7 macrophages, wherein the LCVs were stained with a polyclonal anti-SidC antibody, followed by a Cy5-coupled goat anti-rabbit antibody ( cyan ). Bar, 10 m m. Color fi gure available in the online version of the article (color fi gure online).

31718 Pathogen Vacuole Purifi cation from Legionella-Infected Amoeba and Macrophages

9. Remove the loaded MACS columns from the MACS separator (and thus, the magnetic fi eld), and fi rmly press 0.5 mL ice-cold HS buffer through the column to elute the bound beads linked to LCVs into a 1.5 mL microcentrifuge tube. Set aside 10 m L of the eluate for fl uorescence microscopy (see Note 15; Fig. 2 ).

1. Prepare a linear gradient of 10–35% Histodenz in a total of 11.5 mL PBS. To this end, fi ll 5.75 mL of a 35% Histodenz/PBS solution into a 15 mL capped centrifuge tube and care-fully top with 5.75 mL of a 10% Histodenz/PBS solution without mixing. Cap the tube.

2. Gently lay the tube down horizontally for 1 h and then slowly return it back into a vertical position.

3. Load 0.5 mL of LCVs enriched by immuno-magnetic separa-tion (see Subheading 3.6 ) on top of the 11.5 mL gradient of the 10–35% Histodenz/PBS solution and centrifuge at 3,350 × g for 1 h (4°C).

4. Starting from the bottom of the 15 mL tube holding the 12 mL Histodenz gradient, collect eight 1.5 mL fractions using a Pasteur pipette, and place the fractions on ice. Fraction 1 is at the bottom of the tube. Set aside 30–150 m L of each of the eight Histodenz fractions for fl uorescence microscopy. The fraction 4 is expected to contain the highest concentration of puri fi ed LCVs (Fig. 3 ).

3.7. Puri fi cation of LCVs by Density Gradient Centrifugation

Fig. 3. Legionella -containing vacuoles after density-gradient centrifugation. The eluate from the MACS-column containing intact LCVs was further puri fi ed by density-gradient centrifugation through a linear 10–35% Histodenz gradient, and eight fractions were collected. LCVs containing DsRed-producing L. pneumophila ( red ) are highly enriched in fraction 4 from ( a ) calnexin-GFP producing D. discoideum ( green ) or ( b ) RAW 264.7 macrophages stained with a polyclonal anti-SidC antibody, followed by a Cy5-coupled goat anti-rabbit antibody ( cyan ). Bar, 10 m m. Insets show magni fi cations of the marked area.

318 C. Hoffmann et al.

5. Analyze the puri fi ed LCVs by SDS-PAGE/protein staining, Western blot, immuno- fl uorescence microscopy (see Subheading 3.8 ), and mass spectrometry.

1. Coat a sterile, round microscopy coverslip with sterile 0.01% poly- L -lysine solution, and place it into the well of a 24-well fl at-bottomed tissue culture plate.

2. Add samples containing suspensions of phagocytes infected with DsRed-producing L. pneumophila or cell-free LCVs to the poly- L -lysine-coated coverslips in the wells. Fill the wells containing samples with 1 mL of ice-cold HS buffer to dilute the high Histodenz concentrations and centrifuge at 600 × g for 10 min (4°C).

3. Carefully remove the supernatant, add 0.5 mL/well 4% PFA in PBS, and incubate for 20 min at room temperature.

4. Wash the coverslips twice with 0.5 mL/well SorC buffer ( D. discoideum ) or PBS (RAW 264.7 macrophages). Each time carefully remove the supernatant.

5. LCVs from infected D. discoideum : Mount coverslips on a microscopy glass slide using mounting medium (e.g., Vectashield). Analyze morphology, integrity, and quantity of LCVs per view fi eld with an epi fl uorescence microscope equipped with the required fi lters (Fig. 3 ).

6. LCVs from infected RAW 264.7 macrophages: Block the PFA- fi xed samples with 1% BSA at room temperature for 20 min. Incubate on para fi lm with 30 m L of primary antibody (af fi nity-puri fi ed rabbit anti-SidC; 1:1,000 in blocking buffer) for 1 h. Wash coverslips three times with PBS. Incubate on para fi lm with secondary antibody (e.g., anti-rabbit-Cy5; 1:200 in block-ing buffer) for 1 h. Wash coverslips three times with PBS. Mount coverslips on glass slide using mounting medium (e.g., Vectashield). Analyze morphology, integrity, and quantity of LCVs per view fi eld with an epi fl uorescence microscope equipped with the required fi lters (Fig. 3 ).

1. The source, quality, and composition of yeast extract and peptone affect the virulence of L. pneumophila and the growth of D. discoideum . For a high reproducibility of virulence traits and cell physiology, the additives should be tested and the same suppliers and batches should be used.

2. Dissolve L -cysteine and Fe(NO 3 ) 3 each separately in 10 mL of H 2 O in a 15 mL tube. While stirring slowly fi rst add the

3.8. Analysis of LCV Puri fi cation by Fluorescence Microscopy

4. Notes

31918 Pathogen Vacuole Purifi cation from Legionella-Infected Amoeba and Macrophages

L -cysteine solution, followed by the iron solution to prevent precipitation in the medium.

3. The medium should be pre- fi ltered six to eight times through a glass fi ber fi lter paper to remove precipitates formed while mixing the solution. L -cysteine is light sensitive.

4. Calnexin is an ER-resident protein that also localizes to the LCV membrane ( 22, 25, 27 ) .

5. Glucose caramelizes upon autoclaving in combination with the medium. Suspend 11 g of D (+)glucose in warmed 50 mL of H 2 O, fi lter sterilize with a 0.2 m m fi lter, and add to the auto-claved medium.

6. L. pneumophila bacteria grown to post-exponential/early sta-tionary growth phase in AYE medium are morphologically uniform (~2 × 0.5 m m); i.e., the proportion of long, fi lamentous L. pneumophila (>20 m m) is much smaller than that in bacterial cultures grown on CYE agar plates. The morphology of the bacteria can be easily determined by light microscopy using a small volume (10 m L) of the bacterial culture. The fi nal OD 600 should not be <3.0; otherwise the infection ef fi ciency is severely compromised. L. pneumophila is very sensitive to detergents, and therefore the glassware used should be thoroughly rinsed prior to use.

7. D. discoideum grows axenically (in the absence of bacteria) in a complex medium (e.g., HL5) at 21–23°C. Above 25°C/26°C the amoeba will respond with a heat shock and eventually die. D. discoideum is light sensitive, and therefore frequent alterna-tion between light and dark should be avoided (turn on light in incubator).

8. Prior to the infection, the phagocytes should have reached a con fl uence of about 80%; a higher cell density will negatively affect the uptake ef fi ciency.

9. An OD 600 of 3.0 corresponds to approximately 2 × 10 9 bacte-ria/mL. The exact correlation of the OD 600 with the bacterial concentration depends on the spectrophotometer used and should be determined experimentally.

10. An incubation period of 1 h is the standard infection time for the isolation of LCVs. However, LCVs have been successfully puri fi ed after infections ranging from 15 min up to 14 h ( 14 ) .

11. The protease inhibitor in the HS buffer is essential for RAW 264.7 macrophages, but may be omitted for D. discoideum , with-out signi fi cantly changing the proteome of puri fi ed LCVs ( 14 ) .

12. Use an odd number of strokes to homogenize the suspension of L. pneumophila -infected phagocytes. Thereby, unbroken cells remain in the fi rst syringe and do not end up in the second syringe containing the homogenate to be used further.

320 C. Hoffmann et al.

13. The blocking reagent and concentration should be optimized for each primary antibody. Other reagents such as BSA may be more appropriate for other antibodies.

14. The anti-SidC serum should be suitable to isolate LCVs from different L. pneumophila strains, as long as SidC is abundantly produced and homogenously decorating the LCV membrane. Antibodies against other (bacterial) markers exclusively present on LCV membranes should also work to isolate LCVs.

15. Apply fi rm pressure to elute the column; this will substantially increase the yield of LCVs.

Acknowledgments

This work was supported by the Max von Pettenkofer Institute, Ludwig-Maximilians University Munich, and the German Research Foundation (BMBF “Medical Infection Genomics”, HI 1511/3-1).

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